A novel Superplex RT-qPCR strategy will be optimized for the single-tube amplification and quantification of targeted panels of fusion transcripts that can be broadly applicable for the rapid development of clinical cancer diagnostic assays. The novel design will enable accurate and reproducible quantitative detection of mid-sized RNA panels more than a dozen targets in clinical samples, providing a compact, cost-efficient assay that can be matched to the right clinical need. The approach will address diagnostic laboratories' requirement for reliable, harmonized and affordable methods that are easy to implement and can tap a ubiquitous install base of real-time PCR instruments. The proposed effort will optimize the technology, specifically for fusion transcripts, and incorporate Asuragen's proprietary Armored RNA technology in the final format as stable and versatile RNA controls approved for clinical diagnostics. Together, these innovations will provide a solid foundation for the rapid development of a suite of companion diagnostic assays that can be used as tools for cancer diagnosis and to guide precision medicine options for patients. Superplex RT-qPCR overcomes the limitations of quantitative PCR instruments by increasing the number of targets detected in a single tube through dual readouts of fluorescent intensity and melting temperature to achieve multiplexing in a 2-dimensional solution array. The method combines the use of Molecular Beacon probes (MBs) and an improved version of asymmetric PCR called Linear-after-the-Exponential (LATE) PCR. In Phase I, this method will be optimized and verified with cell lines with documented translocations and using synthetic transcripts spiked in total RNA. The aims are: Specific Aim 1: Develop PCR primers and MB probes for 13 leukemia translocations and one control (14 targets total). Specific Aim 2: Optimize primers and probes for performance in the multiplex reaction. Specific Aim 3: Analytically verify the fully multiplexed assay. Successful completion of phase I will result in a platform on which Asuragen can develop other Superplex assays for hematologic malignancies and leukemia sub?types, including other panels of clinically-actionable cancer fusions such as those in solid tumors. In phase II, we will expand the number of targets detected, incorporate multi-categorical analyte (RNA and DNA) detection, and optimize for manufacturing as a kit. Additionally, RNA standards will be developed to make the kit quantitative and to provide harmonized results across laboratories.